The mechanisms underlying pulmonary hypersensitivity to low molecular mass allergens are uncertain. The objectives of this grant are to elucidate the mechanisms underlying pulmonary hypersensitivity to inhaled chemicals and determine the factors responsible for progression from acute episodic symptoms to chronic allergic lung disease. A guinea pig model (developed by this investigator) is used. Sensitized animals display the features characteristic of chemical sensitivity, i.e., immediate- and late-onset airway responses, airway hyperreactivity to nonspecific agonists, eosinophilic inflammation, inflammatory cytokines, and specific antibodies. Plans for the next 4 years will focus on the mechanisms of chemical sensitivity. The hypothesis to be addressed is that sensitization is initiated by covalent binding of the chemical allergen to particular proteins. The nature of the proteins and the chemistry of hapten-protein binding are critical to the sensitization outcome. Specific Aim 1 is to identify the in vitro chemical adducts and adduction sites of TDI on serum albumin (SA), and on hemoglobin (Hb), two proteins which in guinea pigs have been shown to bear adduct following inhalation of TDI. 14C-labeled 2,4 and 2,6 TDI isomers will be utilized, at varying concentrations, for in vitro exposure of guinea pig SA and Hb. The sites of adduction will be determined by site-specific limited proteolysis followed by LC-MS analyses. Specific Aim 2 is to locate the TDI-adducts formed in vivo using respiratory tissue from both non-sensitized and TDI-sensitized guinea pigs, employing fluorescent labeling techniques. Adducts in the blood of naive and sensitized guinea pigs will be compared with those formed in vitro to assess the presence in sensitized animals of particular TDI-binding macromolecules. Specific Aim 3 is to determine the presence of TDI adducts in blood, bronchoalveolar lavage (BAL) and bronchial biopsies from workers exposed occupationally to TDI to compare the adducts qualitatively and quantitatively, with those identified in vitro and in the guinea pig model. Specific Aim 4 is to determine if the adducted proteins and sites of adduction are unique for TDI, or if they are adducted upon exposure to other industrial isocyanates. Samples from individuals exposed to two other widely used isocyanates, hexamethylene diisocyanate (HDI) and diphenylmethane diisocyanate (MDI), will be analyzed. In this way, comparison can be made of: guinea pig vs. human samples, of adducts formed in naive vs. sensitized animals, and among occupational isocyanates. Specific Aim 5 is to identify adducts involved in the sensitization process using immunoaffinity chromatography, and employing antibodies from guinea pigs (and individuals) sensitized to the chemical. Additionally, these studies will provide biomarkers of isocyanate exposure.